Search

Gastrointestinal mucositis could potentially compromise drug absorption due to functional loss of mucosa and other pathophysiological changes in the gastrointestinal microenvironment. Little is known about this effect on commonly used anti-infectives. This study aimed to explore the association between different stages of gastrointestinal mucositis, drug exposure, and gut microbiota. A prospective, observational pilot study was performed in HSCT patients aged ≥ 18 years receiving anti-infectives orally. Left-over blood samples and fecal swabs were collected from routine clinical care until 14 days after HSCT to analyze drug and citrulline concentrations and to determine the composition of the gut microbiota. 21 patients with a median age of 58 (interquartile range 54-64) years were included with 252 citrulline, 155 ciprofloxacin, 139 fluconazole, and 76 acyclovir concentrations and 48 fecal swabs obtained. Severe gastrointestinal mucositis was observed in all patients. Due to limited data correlation analysis was not done for valacyclovir and fluconazole, however we did observe a weak correlation between ciprofloxacin and citrulline concentrations. This could suggest that underexposure of ciprofloxacin can occur during severe mucositis. A follow-up study using frequent sampling rather than the use of left-over would be required to investigate the relationship between gastrointestinal mucositis, drug exposure, and gut microbiome., (© 2023. The Author(s).)

Background: The use of (val)ganciclovir is complicated by toxicity, slow response to treatment and acquired resistance., Objectives: To evaluate a routine therapeutic drug monitoring (TDM) programme for ganciclovir in a transplant patient population., Methods: An observational study was performed in transplant recipients from June 2018 to February 2020. Dose adjustments were advised by the TDM pharmacist as part of clinical care. For prophylaxis, a trough concentration (Cmin) of 1-2 mg/L and an AUC24h of >50 mg·h/L were aimed for. For treatment, a Cmin of 2-4 mg/L and an AUC24h of 80-120 mg·h/L were aimed for., Results: Ninety-five solid organ and stem cell transplant patients were enrolled. Overall, 450 serum concentrations were measured; with a median of 3 (IQR = 2-6) per patient. The median Cmin and AUC24h in the treatment and prophylaxis groups were 2.0 mg/L and 90 mg·h/L and 0.9 mg/L and 67 mg·h/L, respectively. Significant intra- and inter-patient patient variability was observed. The majority of patients with an estimated glomerular filtration rate of more than 120 mL/min/1.73 m2 and patients on continuous veno-venous haemofiltration showed underexposure. The highest Cmin and AUC24h values were associated with the increase in liver function markers and decline in WBC count as compared with baseline., Conclusions: This study revealed that a standard weight and kidney function-based dosing regimen resulted in highly variable ganciclovir Cmin and under- and over-exposure were observed in patients on dialysis and in patients with increased renal function. Clearly there is a need to explore the impact of concentration-guided dose adjustments in a prospective study., (© The Author(s) 2021. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy.)

An important element in antimicrobial stewardship programmes is early switch from intravenous (i.v.) to oral antimicrobial treatment, especially for highly bioavailable drugs. The antifungal agent voriconazole is available both in i.v. and oral formulations and bioavailability is estimated to be >90% in healthy volunteers, making this drug a suitable candidate for such a transition. Recently, two studies have shown that the bioavailability of voriconazole is substantially lower in patients. However, for both studies various factors that could influence the voriconazole serum concentration, such as inflammation, concomitant intake of food with oral voriconazole, and gastrointestinal complications, were not included in the evaluation. Therefore, in this study a retrospective chart review was performed in adult patients treated with both oral and i.v. voriconazole at the same dose and within a limited (≤5 days) time interval in order to evaluate the effect of switching the route of administration on voriconazole serum concentrations. A total of 13 patients were included. The mean voriconazole trough concentration was 2.28 mg/L [95% confidence interval (CI) 1.29-3.26 mg/L] for i.v. voriconazole administration and 2.04 mg/L (95% CI 0.78-3.30 mg/L) for oral administration. No significant difference was found in the mean oral and i.v. trough concentrations of voriconazole (P = 0.390). The mean bioavailability was 83.0% (95% CI 59.0-107.0%). These findings suggest that factors other than bioavailability may cause the observed difference in voriconazole trough concentrations between oral and i.v. administration in the earlier studies and stress the need for an antimicrobial stewardship team to guide voriconazole dosing., (Copyright © 2016 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.)

Background: During an infection or inflammation, several drug-metabolizing enzymes in the liver are down-regulated, including cytochrome P450 iso-enzymes. Since voriconazole is extensively metabolized by cytochrome P450 iso-enzymes, the metabolism of voriconazole can be influenced during inflammation via reduced clearance of the drug, resulting in higher voriconazole trough concentrations., Objective: To investigate prospectively the influence of inflammation on voriconazole metabolism and voriconazole trough concentrations., Methods: A prospective observational study was performed at the University Medical Center Groningen. Patients were eligible for inclusion if they were ≥18 years old and treated with voriconazole. Voriconazole and voriconazole-N-oxide concentrations were determined in discarded blood samples. To determine the degree of inflammation, C-reactive protein (CRP) concentrations were used. Subsequently, a longitudinal data analysis was performed to assess the effect of inflammation on the metabolic ratio and voriconazole trough concentration., Results: Thirty-four patients were included. In total 489 voriconazole trough concentrations were included in the longitudinal data analysis. This analysis showed that inflammation, reflected by CRP concentrations, significantly influenced the metabolic ratio, voriconazole trough concentration and voriconazole-N-oxide concentration (all P < 0.001), when corrected for other factors that could influence voriconazole metabolism. The metabolic ratio was decreased by 0.99229 N and the voriconazole-N-oxide concentration by 0.99775 N , while the voriconazole trough concentration was increased by 1.005321 N , where N is the difference in CRP units (in mg/L)., Conclusions: This study shows that voriconazole metabolism is decreased during inflammation, resulting in higher voriconazole trough concentrations. Therefore, frequent monitoring of voriconazole serum concentrations is recommended during and following severe inflammation., (© The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Objectives: In patients with invasive aspergillosis (IA), fungal cultures are mostly negative. Consequently, azole resistance often remains undetected. The AsperGenius ® multiplex real-time PCR assay identifies clinically relevant Aspergillus species and four resistance-associated mutations (RAMs; TR34/L98H/T289A/Y121F) in the Cyp51A gene. This multicentre study evaluated the diagnostic performance of this assay on bronchoalveolar lavage (BAL) fluid and correlated the presence of RAMs with azole treatment failure and mortality., Methods: Stored BAL samples from patients with haematological diseases with suspected IA were used. BAL samples that were galactomannan/culture positive were considered positive controls for the presence of Aspergillus. Azole treatment failure and 6 week mortality were compared in patients with and without RAMs that had received ≥5 days of voriconazole monotherapy., Results: Two hundred and one patients each contributed one BAL sample, of which 88 were positive controls and 113 were negative controls. The optimal cycle threshold cut-off value for the Aspergillus species PCR was <38. With this cut-off, the PCR was positive in 74/88 positive controls. The sensitivity, specificity, positive predictive value and negative predictive value were 84%, 80%, 76% and 87%, respectively. 32/74 BAL samples were culture negative. Azole treatment failure was observed in 6/8 patients with a RAM compared with 12/45 patients without RAMs (P = 0.01). Six week mortality was 2.7 times higher in patients with RAMs (50.0% versus 18.6%; P = 0.07)., Conclusions: The AsperGenius ® assay had a good diagnostic performance on BAL and differentiated WT from Aspergillus fumigatus with RAMs, including in culture-negative BAL samples. Most importantly, detection of RAMs was associated with azole treatment failure., (© The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Background: Routine therapeutic drug monitoring of voriconazole seems to be beneficial. This study investigated the therapeutic drug monitoring practices in intensive care to derive possible recommendations for improvement., Methods: A retrospective chart review was performed for patients aged ≥18 years who started treatment with voriconazole, which lasted for at least 3 days while being admitted to an intensive care unit to assess possible differences between the patients with and without voriconazole trough concentrations measured., Results: In 64 (76%) of the 84 patients, voriconazole trough concentrations were measured. The groups differed significantly with respect to the duration of voriconazole treatment and intensive care unit admission. Time of sampling was very early and therefore inappropriate for 49% of the first measured voriconazole trough concentrations and in 48% of the subsequent measured concentrations. Of the 349 trough concentrations measured, 129 (37%) were outside the therapeutic window. In 11% of these cases, no recommendation was provided without identifiable reason. In addition, 27% of recommended dose adjustments were not implemented, probably because the advice was not suited for the specific clinical situation., Conclusions: The performance of voriconazole therapeutic drug monitoring can still be improved although voriconazole concentrations were monitored in most patients. A multidisciplinary approach-for instance by means of antifungal stewardship-will probably be able to overcome problems encountered such as timing of sampling, incompleteness of data in clinical context, and lack of implementation of recommendations.

Background: Leukaemic patients receiving intensive chemotherapy and patients undergoing autologous stem-cell transplantation (ASCT) are routinely screened for oral foci of infection to reduce infectious complications that could occur during therapy. In this prospective study we assessed the effect of leaving chronic oral foci of infection untreated on the development of infectious complications in intensively treated haematological patients., Methods: We included and prospectively evaluated all intensively treated leukaemic patients and patients undergoing ASCT who were referred to our medical centre between September 2012 and May 2014, and who matched the inclusion/exclusion criteria. Acute oral foci of infection were removed before chemotherapy or ASCT, whereas chronic oral foci were left untreated., Results: In total 28 leukaemic and 35 ASCT patients were included. Acute oral foci of infection were found in 2 leukaemic (7%) and 2 ASCT patients (6%), and chronic oral foci of infection in 24 leukaemic (86%) and 22 ASCT patients (63%). Positive blood cultures with microorganisms potentially originating from the oral cavity occurred in 7 patients during treatment, but were uneventful on development of infectious complications., Conclusions: Our prospective study supports the hypothesis that chronic oral foci of infection can be left untreated as this does not increase infectious complications during intensive chemotherapy.

Voriconazole (VCZ) exhibits great inter- and intrapatient variability. The latter variation cannot exclusively be explained by concomitant medications, liver disease or dysfunction, and genetic polymorphisms in cytochrome P450 2C19 (CYP2C19). We hypothesized that inflammatory response in patients under VCZ medication might also influence this fluctuation in concentrations. In this study, we explored the association between inflammation, reflected by the C-reactive protein (CRP) concentration, and VCZ trough concentrations over time. A retrospective analysis of data was performed for patients with more than one steady-state VCZ trough concentration and a CRP concentration measured on the same day. A longitudinal analysis was used for series of observations obtained from many study participants over time. The approach involved inclusion of random effects and autocorrelation in linear models to reflect within-person cross-time correlation. A total of 50 patients were eligible for the study, resulting in 139 observations (paired VCZ and CRP concentrations) for the analysis, ranging from 2 to 6 observations per study participant. Inflammation, marked by the CRP concentration, had a significant association with VCZ trough concentrations (P < 0.001). Covariates such as age and interacting comedication ([es]omeprazole), also showed a significant correlation between VCZ and CRP concentrations (P < 0.05). The intrapatient variation of trough concentrations of VCZ was 1.401 (confidence interval [CI], 0.881 to 2.567), and the interpatient variation was 1.756 (CI, 0.934 to 4.440). The autocorrelation between VCZ trough concentrations at two sequential time points was calculated at 0.71 (CI, 0.51 to 0.92). The inflammatory response appears to play a significant role in the largely unpredictable pharmacokinetics of VCZ, especially in patients with high inflammatory response, as reflected by high CRP concentrations., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Background: Posaconazole exposure seems to be subtherapeutic in some patients with invasive fungal disease. Due to the pharmacokinetic variability of posaconazole, therapeutic drug monitoring may help to optimize the efficacy of this antifungal drug., Methods: A retrospective study of patients treated with posaconazole from January 2008 to April 2014 and for whom posaconazole serum concentrations were available was conducted. Risk factors for underexposure of posaconazole were detected, and the relationship between posaconazole exposure and treatment outcome according to the European Organization for Research and Treatment of Cancer (EORTC) criteria was assessed., Results: Seventy patients met the inclusion criteria, 45 patients received posaconazole as treatment, and 25 patients received posaconazole as a prophylactic. Posaconazole serum trough concentrations were <1.25 mg/L in 44.4% of patients receiving treatment and <0.7 mg/L in 40.0% of patients receiving prophylactic posaconazole. Multiple linear regression analysis showed a significant, independent, and negative association of the posaconazole serum trough concentration with a lack of enteral nutrition (P < 0.001), vomiting (P = 0.035), the use of a proton pump inhibitor or H2-receptor antagonist (P < 0.001), a liquid diet (P = 0.002), concomitant chemotherapy (P = 0.004), and a posaconazole dose frequency of 2 times daily (P = 0.015). A higher posaconazole concentration was associated with a better treatment outcome [odds ratio = 22.22 (95% confidence interval, 3.40-145.33); P = 0.001]., Conclusions: Posaconazole exposure is insufficient in more than 40% of patients at risk of or with invasive fungal disease, and posaconazole exposure is positively correlated with a successful treatment outcome. Therapeutic drug monitoring of posaconazole can detect underexposure and can be helpful in treatment optimization.

Voriconazole pharmacokinetics shows a large inter- and intrapatient variability. Inflammation is associated with changes in the expression of CYP isoenzymes. Here, we evaluated the influence of inflammation, marked by C-reactive protein (CRP) levels in blood, on the metabolism of voriconazole. Observational data showed an association between CRP level and the ratio of voriconazole N-oxide to voriconazole., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Voriconazole concentrations display a large variability, which cannot completely be explained by known factors. Inflammation may be a contributing factor, as inflammatory stimuli can change the activities and expression levels of cytochrome P450 isoenzymes. We explored the correlation between inflammation, reflected by C-reactive protein (CRP) concentrations, and voriconazole trough concentrations. A retrospective chart review of patients with at least one steady-state voriconazole trough concentration and a CRP concentration measured on the same day was performed. A total of 128 patients were included. A significantly (P < 0.001) higher voriconazole trough concentration was observed in patients with severe inflammation (6.2 mg/liter; interquartile range [IQR], 3.4 to 8.7 mg/liter; n = 20) than in patients with moderate inflammation (3.4 mg/liter; IQR, 1.6 to 5.4 mg/liter; n = 60) and in patients with no to mild inflammation (1.6 mg/liter; IQR, 0.8 to 3.0 mg/liter; n = 48). The patients in all three groups received similar voriconazole doses based on mg/kg body weight (P = 0.368). Linear regression analyses, both unadjusted and adjusted for covariates of gender, age, dose, route of administration, liver enzymes, and interacting coadministered medications, showed a significant association between voriconazole and CRP concentration (P < 0.001). For every 1-mg/liter increase in the CRP concentration, the voriconazole trough concentration increased by 0.015 mg/liter (unadjusted 95% confidence interval [CI], 0.011 to 0.020 mg/liter; adjusted 95% CI, 0.011 to 0.019 mg/liter). Inflammation, reflected by the C-reactive protein concentration, is associated with voriconazole trough concentrations. Further research is necessary to assess if taking the inflammatory status of a patient into account is helpful in therapeutic drug monitoring of voriconazole to maintain concentrations in the therapeutic window, thereby possibly preventing suboptimal treatment or adverse events., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Invasive aspergillosis and candidemia are important causes of morbidity and mortality in immunocompromised and critically ill patients. The triazoles voriconazole, fluconazole, and posaconazole are widely used for the treatment and prophylaxis of these fungal infections. Due to the variability of the pharmacokinetics of the triazoles among and within individual patients, therapeutic drug monitoring is important for optimizing the efficacy and safety of antifungal treatment. A dried blood spot (DBS) analysis was developed and was clinically validated for voriconazole, fluconazole, and posaconazole in 28 patients. Furthermore, a questionnaire was administered to evaluate the patients' opinions of the sampling method. The DBS analytical method showed linearity over the concentration range measured for all triazoles. Results for accuracy and precision were within accepted ranges; samples were stable at room temperature for at least 12 days; and different hematocrit values and blood spot volumes had no significant influence. The ratio of the drug concentration in DBS samples to that in plasma was 1.0 for voriconazole and fluconazole and 0.9 for posaconazole. Sixty percent of the patients preferred DBS analysis as a sampling method; 15% preferred venous blood sampling; and 25% had no preferred method. There was significantly less perception of pain with the DBS sampling method (P = 0.021). In conclusion, DBS analysis is a reliable alternative to venous blood sampling and can be used for therapeutic drug monitoring of voriconazole, fluconazole, and posaconazole. Patients were satisfied with DBS sampling and had less pain than with venous sampling. Most patients preferred DBS sampling to venous blood sampling.

Introduction: Nowadays Enterococcus faecium has become one of the most emerging and challenging nosocomial pathogens. The aim of this study was to determine risk factors in haematology patients who are at risk of an Enterococcus faecium bloodstream infection (BSI) and should be considered for pre-emptive glycopeptide treatment. With these identified risk factors a prediction model can be developed for clinical use., Methods: Retrospectively clinical and microbiological data in 33 patients with an E. faecium BSI were compared to 66 control patients during a 5-year period at the haematology ward. Multivariate logistic regression was used to explore the independent risk factors and a prediction model was developed to determine the risk of an E. faecium BSI., Results: E. faecium BSIs were found to be associated with high mortality rates. Independent risk factors for E. faecium BSI were colonization with E. faecium 30 days prior to blood culture (OR 5.71; CI 1.7-18.7), combination of neutropenia and abdominal focus (4.37; 1.4-13.4), age > 58 years (4.01; 1.3-12.5), hospital stay prior to blood culture > 14 days (3.55; 0.98-12.9) and CRP (C-reactive protein) level >125 mg/L (4.37; 1.1-10.2)., Conclusion: Using data from this study, risk stratification for the development of an E. faecium BSI in patients with haematological malignancies is possible. Pre-emptive treatment should be considered in those patients who are at high risk. Using a prediction model as designed in this study, antibiotic stewardship in terms of prudent use of glycopeptides can be improved and might be helpful in controlling further spread of VRE (vancomycin resistant enterococci).

Background: The prognosis of patients with haematological malignancies who are admitted to the ICU is generally poor. In order to optimize care, it is important to be able to determine which patients are most likely to benefit from continuation of treatment after ICU admission., Methods: Data of 86 patients with a haematological malignancy consecutively admitted to the ICU of Maastricht University Medical Centre were examined in a retrospective cohort study in order to identify clinically useful prognostic parameters., Results: ICU mortality was 56% and in-hospital mortality was 65%. Non-survivors had higher APACHE-II and SOFA scores compared with survivors (32±8.0 versus 25±6.5 and 11.5±3.1 versus 8.5±3.0, respectively). The mortality rate was significantly higher in patients with an increasing SOFA score (≥2 points) compared with patients with an unchanged or decreasing SOFA score (72% versus 58% and 21%, respectively). Mortality was also higher in patients requiring invasive mechanical ventilation or inotropic/vasopressor therapy., Conclusion: The mortality rate among patients with haematological malignancies who are admitted to the ICU is high and mainly associated with the severity of illness, as reflected by more severe and worsening organ failure and a need for mechanical ventilation or inotropic/vasopressor therapy. Several factors appear to be associated with a poor outcome, but no absolute predictors of mortality could be identified, although the results suggest that changes in the SOFA score during the stay in the ICU can be helpful in the decision making about the continuation or discontinuation of treatment., (Copyright © 2010 European Federation of Internal Medicine. Published by Elsevier B.V. All rights reserved.)

The objective of this study was to evaluate the cost-effectiveness of posaconazole versus fluconazole for the prevention of invasive fungal infections (IFI) in graft-versus-host disease (GVHD) patients in the Netherlands. A decision analytic model was developed based on a double-blind randomized trial that compared posaconazole with fluconazole antifungal prophylaxis in recipients of allogeneic HSCT with GVHD who were receiving immunosuppressive therapy (Ullmann et al., N Engl J Med 356:335-347, 2007). Clinical events were modeled with chance nodes reflecting probabilities of IFIs, IFI-related death, and death from other causes. Data on life expectancy, quality-of-life, medical resource consumption, and costs were obtained from the literature. The total cost with posaconazole amounted to 9,428 (95% uncertainty interval 7,743-11,388), which is 4,566 (2,460-6,854) more than those with fluconazole. Posaconazole prophylaxis resulted in 0.17 (0.02-0.36) quality adjusted life year (QALY) gained compared to fluconazole prophylaxis, corresponding to an incremental cost effectiveness ratio (ICER) of 26,225 per QALY gained. A scenario analysis demonstrated that at an increased background IFI risk (from 9% to 15%) the ICER was 13,462 per QALY. Given the underlying data and assumptions, posaconazole prophylaxis is expected to be cost-effective relative to fluconazole in recipients of allogeneic HSCT developing GVHD in the Netherlands. The cost-effectiveness of posaconazole depends on the IFI risk, which can vary by hospital.

Background: The aetiology of central nervous system lesions observed in cerebral cyclosporine neurotoxicity remains controversial., Case Presentation: We report a 48-year-old woman with a non-severe aplastic anaemia who presented with stroke-like episodes while on cyclosporine treatment.Transcranial Doppler ultrasound revealed severely elevated flow velocities in several cerebral vessels, consistent with vasospasm. Immediately after reducing the cyclosporine dose, the stroke-like episodes disappeared. Only after cyclosporine withdrawal the transcranial Doppler ultrasound abnormalities fully resolved., Conclusions: This case demonstrates a significant role of vasospasm in the pathway of cyclosporine-induced neurotoxicity. Transcranial Doppler ultrasound is an effective tool for the diagnosis and follow-up of cyclosporine-induced vasospasm.

Background: Acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) patients experience prolonged neutropenia after treatment with intensive chemotherapy, leading to a high risk of invasive fungal infections (IFI). The present study evaluates the cost effectiveness of posaconazole vs. standard azoles for the prevention of IFIs in neutropenic patients in the Netherlands., Methods: A decision-tree model was developed using data from a randomized trial that compared posaconazole and standard azole (fluconazole or itraconazole) prophylaxis in neutropenic patients receiving remission-induction chemotherapy for AML/MDS (Cornely et al., N Engl J Med 2007;356:348-359). Following initiation of prophylaxis, clinical events are modeled with chance nodes reflecting probabilities of IFIs, IFI-related death, and death from other causes. Patients surviving the prophylaxis are assumed to have a life expectancy according to the underlying condition. This allows translation of the trial outcomes to a lifetime horizon. Data on life expectancy, quality of life, medical resource consumption and costs were obtained from the literature. Model outcomes include cost per life year (LY) gained and cost per quality adjusted life year (QALY) gained., Results: The total cost (treatment of breakthrough IFI + prophylaxis) for posaconazole amounted to 4412 euros (95% uncertainty interval 3403 euros - 5666 euros), which is -183 euros (-1985 euros to 1564 euros) less than costs with standard azoles. Posaconazole prophylaxis resulted in 0.08 (0.02-0.15) QALYs gained in comparison with prophylaxis with standard azoles. Results from a probabilistic sensitivity analysis indicate that there is a 90% probability that the cost per QALY gained with posaconazole is below 20,000 euros. Additional scenario analyzes with different assumptions confirmed these findings., Conclusion: Given the underlying data and assumptions, the economic evaluation demonstrated that posaconazole prophylaxis is expected to be cost-effective compared with fluconazole/itraconazole in neutropenic AML/MDS patients after intensive chemotherapy.

Enhanced proliferation of MDS progenitors is abrogated by increased apoptosis of their progeny in vivo. We investigated whether bone marrow mononuclear cells (BMMNC) of MDS patients also showed enhanced proliferation and apoptosis in vitro in comparison with acute myeloid leukemia (AML) and normal BM (NBM). NBM showed a decrease in the number of clusters in time due to apoptosis of clusters and due to development of clusters into colonies with low apoptotic level. In MDS patients, about two-fold more clusters have developed at day 4, and in contrast with NBM, the total number of clusters at day 7 remained high in spite of an increasing percentage of apoptotic clusters (from 52 to 76%) in combination with more colony formation. The number of clusters and colonies showed a sharp decrease at day 10 because of persistently high apoptosis at cluster level and increasing apoptosis in colonies. BMMNC of AML patients showed a decreased proliferation with enhanced apoptosis at cluster level in contrast to a relatively low apoptotic levels in the colony-forming cells. This data show that increased proliferation is abrogated by enhanced apoptosis in MDS, whereas AML showed decreased proliferation with a low level of apoptosis in colony-forming cells. These growth profiles of BMMNC are independent of stromal influences and may represent intrinsic features of the MDS progenitors and accessory cell interactions.

Objective: Bone marrows (BM) of myelodysplastic syndrome (MDS) patients show increased proliferation and premature programmed cell death (PCD) in vivo as well as in vitro. We explored the proliferative capacity and apoptotic propensity of CD34+ progenitor cells of MDS patients excluding accessory cell interference., Materials and Methods: CD34+/CD3-/CD19- cells of 5 MDS patients and 5 normal BM were sorted as single cells into single wells and were cultured in liquid medium. Wells were evaluated on days 4, 7, 10, and 14. PCD was determined by staining with annexin V-FITC. Growth rate and cell doubling time (Td) were calculated for each colony-forming cell., Results: Normal BM CD34+ cells formed clusters and colonies and both showed increasing PCD in time, although within colonies the degree of apoptosis was twice as high (about 25%) as compared with clusters at all time points. In MDS increased cluster formation was observed at all evaluation points when compared to normal BM, whereas the number of colonies was markedly reduced (1/7 of normal). These colonies were also smaller, usually smaller than 100 cells. Significantly enhanced levels of PCD of clusters (53-79%) in combination with longer cell doubling times explain this slower formation of smaller colonies. Surprisingly, these colonies showed considerably lower levels of PCD (7-32%) as compared to normal (1-48%, median values)., Conclusions: In the absence of stromal influences and accessory cells, this study in MDS patients showed intrinsically enhanced proliferation and apoptosis of cluster-forming cells, as the opposite was true for colony-forming cells.

In this paper a patient with a non-Hodgkin's lymphoma (NHL) and paraneoplastic pemphigus (PNP) is described. PNP is a very rare, painful mucocutaneous intraepithelial blistering disease associated with occult or confirmed malignancy. Patients with PNP show severe, progressive mucocutaneous disease with a high mortality rate, because of drug-induced infectious complications. The patients sometimes benefit from high doses of oral corticosteroids. However, pulse therapy with high doses of prednisolone (or dexamethasone) in combination with other immunosuppressants induces variable and inconstant results. Intravenous immunoglobulin (IVIg) has been applied in different cases of PNP with encouraging results. Plasmapheresis or plasma exchange (PE) in combination with corticosteroids and/or cyclophosphamide or azathioprine showed similar rapid and beneficial results in association with decreasing auto-antibody levels in this group of refractory pemphigus. Another interesting therapeutic option is rituximab, a chimeric monoclonal antibody directed against the CD20 antigen, which is found on the surface of normal and malignant B-lymphocytes. Administration of rituximab for patients with PNP in combination with follicular NHL is not always successful regarding oral lesions as we report in this case. PE leading to prompt depletion of autoreactive antibodies combined with immunosuppressants or synchronisation of PE with IVIg seems the best treatment modality for this refractory group, but the therapeutic value and appropriate timing of rituximab obviously deserve further evaluation in patients with low grade NHL and PNP.

Background: To study the apoptotic process in time, we used the following flow cytometric (FCM) techniques: phosphatidylserine (PS) translocation by Annexin-V (AnV), DNA fragmentation by in situ end labeling (ISEL), and propidium iodide (PI) staining. Because PS translocation is assumed to be an early feature of programmed cell death (PCD), we questioned if AnV positivity implies inevitable cell death., Methods: Apoptosis was induced in Jurkat cells by gamma-irradiation, incubation with camptothecin (CPT), or cytosine beta-D-arabinofuranoside (Ara-C). At different time intervals, PCD was quantified by AnV/PI and ISEL. To analyze the influence of cell handling procedures on PCD, we applied these three FCM techniques on CD34+ bone marrow (BM) stem cells after selection and after a freeze-thaw procedure. Various AnV/PI- CD34+ fractions were cultured in a single-cell single-well (SCSW) assay., Results: Jurkat cells under three different detrimental conditions showed essentially the same pattern of apoptosis in time. Initially developed AnV+/PI- cells subsequently (within 1 h) showed ISEL positivity, after which they turned into AnV+/PI++ cells with even higher levels of ISEL positivity (80-90%). Eventually, they lost some of their PI and ISEL positivity and formed the AnV+/PI+ fraction. Cell handling of CD34+ cells caused high and variable AnV+/PI- fractions (overall range 23-62%). Within total AnV+ and AnV+/PI- populations, only a minority of CD34+ cells showed ISEL positivity (range 4-8% and 0.8-6%, respectively). Different fractions of AnV+/PI- CD34+ cells did have clonogenic capacity., Conclusions: PCD of cell suspensions in vitro can be followed accurately in time by these three FCM techniques. PS translocation is followed rapidly (within 1 h) by oligo-nucleosomal DNA fragmentation, after which cell (and nuclear) membrane leakage occurs. Detection of PS asymmetry by AnV-fluorescein isothiocyanate (FITC) is not always associated with (inevitable) apoptosis, as can be concluded from the proliferative capacity of AnV+ /PI- CD34+ cells in the SCSW assay., (Copyright 2001 Wiley-Liss, Inc.)

Myelodysplastic syndromes (MDS) are highly proliferative bone marrow (BM) disorders where the primary lesion presumably affects a CD34+ early progenitor or stem cell. We investigated the proliferative characteristics of CD34+ cells of 33 untreated MDS patients (19 RA, 5 RARS, 7 RAEB, 2 RAEBt) and five patients with acute myeloid leukemia after MDS (sAML). All patients received a 1-h infusion of the thymidine analogue iodoor bromodeoxyuridine intravenously before a BM aspirate and biopsy was taken. A double-labeling immunohistochemistry technique by monoclonal anti-CD34 (QBend/10) and anti-IUdR/BrdU antibodies was developed and performed. By this technique we recognised CD34+ and CD34- cells actively engaged in DNA synthesis or not. As MDS evolves a significant increase occurred in the percentage of CD34+ cells of all myeloid cells (mean value: RA/RARS 1.67%; RAEB(t) 8.68%; sAML 23.83%) as well as in the percentage of proliferating CD34+ cells of all myeloid cells (RA/RARS 0.19%; RAEB(t) 0.43%; and sAML 3.30%). This was associated with a decreasing trend in the overall myeloid labeling index (LI: RA/RARS 25.8%, RAEB(t) 24.6% and sAML 21.5%). This decrease in overall myeloid LI is due to an exponential increase in the proportion of CD34+ cells of the proliferating compartment during MDS evolution (RA/RARS 0.35%, RAEB(t) 1.44% and sAML 11.98% of all S-phase cells). These CD34+ cells appeared to proliferate more slowly than their more mature CD34 negative counterparts, since we found a progressive increment in the mean total cell cycling time (Tc) of all myeloid cells during MDS progression (RA/RARS 39.8, RAEB(t) 45.2 and sAML 65.8 h). This study showed that during MDS evolution to sAML the CD34+ compartment develops a growth advantage leading to apparent expansion.

The present study describes the effect of plasma exchange or whole blood exchange (PEBE) on the survival rate among patients with fulminant meningococcal sepsis and on the level of circulating endotoxin. Since 1989 all patients with meningococcal disease and hypotension who were admitted to our intensive care unit were treated with PEBE. Results for our patients were compared with those for a historical control group conventionally treated between 1984 and 1989 (n = 10; mortality rate, 60%); the expected mortality rate, which was based on the Niklasson prognostic score and was calculated for seven patients in this control group, was 73%. A total of 15 patients were treated with PEBE, three (20%) of whom died, whereas the prognostic score (calculated for 14 patients) for this group was 62%. In two of the fatal cases, PEBE was started after a delay of greater than or equal to 40 hours. In the remaining 13 patients, PEBE was started within 5-30 hours after the first hospital admission. The mortality rate among this group was 8% (one of 13 patients); this rate was significantly different from that among the control group (P = .025). For seven patients treated with PEBE, plasma endotoxin concentrations were sequentially measured. The overall half-life (+/- SEM) of endotoxin was 181 +/- 18 minutes. This is approximately the same as reported values for patients who were not treated with PEBE. It is concluded that early initiation of PEBE may improve the rate of survival among patients with meningococcal infection and hypotension but that the mechanism of the beneficial effect is most likely not based on the elimination of endotoxin.

Plasma cortisol levels and modified Apache II (Apache IIm-stay) severity of disease scores were determined at weekly intervals in 159 patients who were treated for at least 7 days at the Critical Care Unit of our hospital. The mean (+/- SD) plasma cortisol level (0.60 +/- 0.28 mumol/l) was clearly elevated in these patients. The highest plasma cortisol levels were measured in patients treated with vasoactive drugs (0.76 +/- 0.39 mumol/l). Non-survivors (n = 36) had a significantly higher mean plasma cortisol level and Apache IIm-stay score than survivors (respectively 0.78 +/- 0.40 vs. 0.54 +/- 0.21 mumol/l; p less than 0.0003 and 12.6 +/- 4.8 vs. 7.3 +/- 4.1; p less than 0.0001). A significant correlation was found between the individual weekly plasma cortisol levels and the Apache IIm-stay scores (r = 0.41; p less than 0.0001), especially in the subgroup of patients, who never received glucocorticoids during their stay at the ICU (r = 0.51; p less than 0.0001). During the 14-month study period only two patients showed a clinical picture of adrenocortical insufficiency and a blunted response of cortisol to 0.25 mg synthetic ACTH(1-24). In conclusion, our data suggest that a high plasma cortisol level, like a high Apache IIm-stay score, indicates severity of disease and poor survival in critically ill patients. De novo adrenocortical insufficiency is rare and therefore routine screening of adrenocortical function is superfluous.